As recombinant DNA biotechnology has developed in recent years, the controlled production by microorganisms of an enormous variety of useful polypeptides has become possible. Many polypeptides, such as for example human growth hormone, leukocyte interferons, human insulin, and human proinsulin liave already been produced by various microorganisms. The continued application of techniques already in hand is expected to permit production of a variety of other useful polypeptide products.
The basic techniques employed in the field of recombinant DNA technology are known by those of skill in the art. The elements desirably present for the practice of recombinant DNA technology include but are not limited to:
(1) a gene coding for one or more desired polypeptide(s) and functionally associated with adequate control sequences required for expression of the gene in the bost organism; PA1 (2) a vector into which the gene can be inserted; PA1 (3) a suitable bost organism into which the vector carrying the gene can be transformed; PA1 (4) if secretion of the heterologous protein is desired, a signal sequence capable of directing the heterologous protein into the secretion pathway of the host cell, and thereafter out of the cell; PA1 (5) a transformation system; and PA1 (6) a method of selecting transformants.
Recombinant gene constructs can be designed such that the recombinant protein transits the host's secretory pathway and is secreted into the growth media. Secretion is a desired mode of recombinant expression for several reasons. First, some heterologous proteins have a toxic effect on ttie bost organism. When such heterologous gene products are secreted rather than accumulated within the host, they are less likely to interfere with normal cellular functions. Second, some proteins that are inactive when produced intracellularly are active when secreted. Third, secretion into the medium avoids the necessity of breaking open the host cells in order to recover the product. Product purification is much easier and cost effective when product is present in the growth medium. And, fourth, since the recombinant product is present in the nutrient medium, the desired product can be continuously removed and the media can be recycled.
Most secreted proteins are expressed initially inside the cell in a precursor or a pre-protein form, containing an appended amino terminal extension called a signal peptide. The signal peptide plays an essential role in transporting the appended polypeptide into and/or through the limiting cellular membranes. This signal peptide is then cleaved proteolytically by a signal peptidase during or after secretion to yield a mature protein product.
Secretion of a heterologous or foreign protein can be accomplished by linking the coding sequence of the heterologous DNA to DNA encoding a signal peptide. It would be desirable to isolate a signal sequence encoding this signal peptide, which would facilitate secretion.
Signal sequences are especially useful in the creation of expression vectors. The use of such vectors would make it possible to transform compatible host cells so that they produce and secrete heterologous gene products. Examples of leader sequences which have been used to successfully secrete recombinant proteins from yeast bosts include those from the Saccbaromyces cerevisiae alpha mating factor a mating factor, and killer toxin genes. Isolation of a signal sequence from a methylotrophic yeast, such as Pichia pastoris, has not been described.
Conveniently, the promoter which is employed in such vectors to regulate expression of the heterologous gene products may be the promoter natively associated with the signal sequence. It would be especially advantageous if the promoter natively associated with the signal sequence provides for a high level of DNA transcription and is responsive to exogenous environmental stimuli. An example of such a promoter is the 5' regulatory region of the Pichia pastoris acid phosphatase (PHO1) gene (SEQ ID NO:3), which is transcribed at a high level in response to the absence of phosphate in the media, and repressed by the presence of phosphate in the media.
It is often desirable to transform a Pichia pastoris host with a recombinant DNA construct that will integrate at a precise position in the Pichia pastoris genome. The 5' and 3' sequences which flank the Pichia pastoris PHO1 gene, also known as first and second insertable DNA fragments, respectively, are used in expression vectors to direct the integration of the recombinant sequences at the PHO1 locus. The ability to integrate recombinant DNAs at the PHO1 locus is advantageous for at least two reasons: 1) in the development of Pichia pastoris expression strains having multiple copies of the same or different expression cassettes at the PHO1 locus or another Pichia locus, or 2) stable integration of one or more expression cassettes at the PHO1 locus only, in a host Pichia pastoris strain wherein disruption of an essential gene or a gene of the methanol metabolism pathway is undesirable.
Cells in which the PHO1 gene has been disrupted show a concomitant loss of acid phosphatase enzyme activity. The Pho.sup.- phenotype, indicative of PHO1 gene disruption, may be screened for by plating the cells on low phosphate indicator plates and allowing colonies to grow overnight. Colonies in which the PHO1 gene is disrupted are white, whereas those colonies having an intact PHO1 gene are green. This colorimetric screen provides a rapid and easy method for detecting cells which have integrated expression cassettes correctly at the PHO1 locus and thus disrupted it.
Thus, it would be a significant contribution to the art to isolate a signal sequence that would facilitate the secretion of proteins from a host cell.
Additionally, it would be advantageous to isolate a 5' regulatory region which would provide for high levels of DNA transcription and is responsive to exogenous environmental stimuli. Currently no 5' regulatory region is known in the art which is transcribed at a high level in response to the absence of phosphate in the media and which can be used with the higbly productive fermentation yeast Pichia pastoris.
It would additionally be advantageous to isolate the acid phosphatase (AP) structural gene.
It would also be advantageous to provide novel vectors comprising fragments of the acid phosphatase gene.
It would additionally be advantageous to isolate a 3' transcription termination sequence.
It would also be advantageous to provide integrative vectors which would direct integration at the Pichia pastoris PHO1 locus.
It would additionally be advantageous to provide a method of identifying disruptants.
Thus, it is an object of the present invention to provide a signal sequence which facilitates the secretion of proteins from cells.
It is also an object of this invention to provide a 5' regulatory region transcribed in response to the absence of phosphate.
Another object of the present invention is to provide the DNA sequence of the Pichia pastoris acid phosphatase structural gene (SEQ ID NO:6).
It is a further object of this invention to provide novel vectors comprising a regulatory region and/or signal sequence operably-linked to a heterologous DNA sequence which encodes at least one polypeptide, and means of inducing said regulatory region to facilitate expression of said heterologous DNA sequence.
It is a still further object of this invention to provide a 3' transcription termination sequence from the acid phosphatase gene.
Yet another object of this invention is to provide integrative vectors which would direct integration at the Pichia pastoris PHO1 locus.
A further object of this invention is to provide a method of identifying disruptants.
Other aspects, objects, and advantages of the present invention will become apparent from the following specification, examples, and claims.